Jon D. Stewart

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Name: Stewart, Jon D.

Organization: University of Florida , USA

Department: Department of Chemistry

Title: Professor(PhD)

TOPICS

Organic Chemistry

Polymer

Conductive Polymers RAFT

Chemicals
References

An Efficient Chemoenzymatic Synthesis of Coenzyme A and Its Disulfide

Co-reporter:Louis M. M. Mouterde and Jon D. Stewart

Organic Process Research & Development 2016 Volume 20(Issue 5) pp:954-959

Publication Date(Web):April 8, 2016

DOI:10.1021/acs.oprd.6b00059

We have developed a chemoenzymatic route to coenzyme A (CoASH) and its disulfide that is amenable to gram-scale synthesis using standard laboratory equipment. By synthesizing the symmetrical disulfide of pantetheine (pantethine), we avoided the need to mask the reactive sulfhydryl and also prevented sulfur oxidation byproducts. No chromatography is required in our synthetic route to pantethine, which facilitates scale-up. Furthermore, we discovered that all three enzymes of the CoASH salvage pathway (pantetheine kinase, phosphopantetheine adenyltransferase, and dephospho-coenzyme A kinase) accept the disulfide of the natural substrates and functionalize both ends of the molecules. This yields CoA disulfide as the product of the enzymatic cascade, a much more stable form of the cofactor. Free CoASH can be prepared by in situ S–S reduction.

Role of Polymer Architecture on the Activity of Polymer–Protein Conjugates for the Treatment of Accelerated Bone Loss Disorders

Co-reporter:Bryan S. Tucker, Jon D. Stewart, J. Ignacio Aguirre, L. Shannon Holliday, C. Adrian Figg, Jonathan G. Messer, and Brent S. Sumerlin

Biomacromolecules 2015 Volume 16(Issue 8) pp:

Publication Date(Web):July 7, 2015

DOI:10.1021/acs.biomac.5b00623

Polymers of similar molecular weights and chemical constitution but varying in their macromolecular architectures were conjugated to osteoprotegerin (OPG) to determine the effect of polymer topology on protein activity in vitro and in vivo. OPG is a protein that inhibits bone resorption by preventing the formation of mature osteoclasts from the osteoclast precursor cell. Accelerated bone loss disorders, such as osteoporosis, rheumatoid arthritis, and metastatic bone disease, occur as a result of increased osteoclastogenesis, leading to the severe weakening of the bone. OPG has shown promise as a treatment in bone disorders; however, it is rapidly cleared from circulation through rapid liver uptake, and frequent, high doses of the protein are necessary to achieve a therapeutic benefit. We aimed to improve the effectiveness of OPG by creating OPG–polymer bioconjugates, employing reversible addition–fragmentation chain transfer polymerization to create well-defined polymers with branching densities varying from linear, loosely branched to densely branched. Polymers with each of these architectures were conjugated to OPG using a “grafting-to” approach, and the bioconjugates were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The OPG–polymer bioconjugates showed retention of activity in vitro against osteoclasts, and each bioconjugate was shown to be nontoxic. Preliminary in vivo studies further supported the nontoxic characteristics of the bioconjugates, and measurement of the bone mineral density in rats 7 days post-treatment via peripheral quantitative computed tomography suggested a slight increase in bone mineral density after administration of the loosely branched OPG–polymer bioconjugate.

Residues Controlling Facial Selectivity in an Alkene Reductase and Semirational Alterations to Create Stereocomplementary Variants

Co-reporter:Adam Z. Walton, Bradford Sullivan, Athéna C. Patterson-Orazem, and Jon D. Stewart

ACS Catalysis 2014 Volume 4(Issue 7) pp:2307

Publication Date(Web):May 30, 2014

DOI:10.1021/cs500429k

A systematic saturation mutagenesis campaign was carried out on an alkene reductase from Pichia stipitis (OYE 2.6) to develop variants with reversed stereoselectivities. Wild-type OYE 2.6 reduces three representative Baylis–Hillman adducts to the corresponding S products with almost complete stereoselectivities and good catalytic efficiencies. We created and screened 13 first-generation, site-saturation mutagenesis libraries, targeting residues found near the bound substrate. One variant (Tyr78Trp) showed high R selectivity toward one of the three substrates, but no change (cyclohexenone derivative) and no catalytic activity (acrylate derivative) for the other two. Subsequent rounds of mutagenesis retained the Tyr78Trp mutation and explored other residues that impacted stereoselectivity when altered in a wild-type background. These efforts yielded double and triple mutants that possessed inverted stereoselectivities for two of the three substrates (conversions >99% and at least 91% ee (R)). To understand the reasons underlying the stereochemical changes, we solved crystal structures of two key mutants: Tyr78Trp and Tyr78Trp/Ile113Cys, the latter with substrate partially occupying the active site. By combining these experimental data with modeling studies, we have proposed a rationale that explains the impacts of the most useful mutations.Keywords: alkene reductase; directed evolution; mutagenesis; old yellow enzyme; protein engineering; X-ray crystallography

Pichia stipitis OYE 2.6 variants with improved catalytic efficiencies from site-saturation mutagenesis libraries

Co-reporter:Athéna Patterson-Orazem, Bradford Sullivan, Jon D. Stewart

Bioorganic & Medicinal Chemistry 2014 Volume 22(Issue 20) pp:5628-5632

Publication Date(Web):15 October 2014

DOI:10.1016/j.bmc.2014.07.001

Influence of Cofactor Regeneration Strategies on Preparative-Scale, Asymmetric Carbonyl Reductions by Engineered Escherichia coli

Co-reporter:Dimitri Dascier, Spiros Kambourakis, Ling Hua, J. David Rozzell, and Jon D. Stewart

Organic Process Research & Development 2014 Volume 18(Issue 6) pp:793-800

Publication Date(Web):February 17, 2014

DOI:10.1021/op400312n

This study was designed to determine whether whole cells or crude enzyme extracts are more effective for preparative-scale ketone reductions by dehydrogenases as well as learning which cofactor regeneration scheme is most effective. Based on results from three representative ketone substrates (an α-fluoro-β-keto ester, a bis-trifluoromethylated acetophenone, and a symmetrical β-diketone), our results demonstrate that several nicotinamide cofactor regeneration strategies can be applied to preparative-scale dehydrogenase-catalyzed reactions successfully.

X-ray Crystallography Reveals How Subtle Changes Control the Orientation of Substrate Binding in an Alkene Reductase

Co-reporter:Yuri A. Pompeu, Bradford Sullivan, and Jon D. Stewart

ACS Catalysis 2013 Volume 3(Issue 10) pp:2376

Publication Date(Web):September 9, 2013

DOI:10.1021/cs400622e

Reductions of (S)- and (R)-carvone by wild-type Saccharomyces pastorianus Old Yellow Enzyme (OYE 1) and a systematic collection of Trp 116 variants revealed that, for (S)-carvone, six Trp 116 mutants displayed inverted diastereoselectivity compared to the wild-type. For example, Ile and Val showed inverted stereoselectivity, but Leu and Phe maintained the wild-type stereopreference. For (R)-carvone, only two Trp 116 mutants (Ala and Val) reduced this alkene with reversed selectivity; all other catalytically active variants including Leu and Ile retained the wild-type diastereoselectivity. The same set of mutant enzymes was also used to catalyze the dehydrogenation of (S)- and (R)-carvone under aerobic conditions. To understand how small changes to the active site structure of OYE 1 could significantly influence its catalytic properties, we solved X-ray crystal structures of the wild-type as well as six key Trp 116 variants after individually soaking with both (S)- and (R)-carvone. In many cases, pseudo-Michaelis complexes formed in crystallo, and these revealed the details of protein–substrate interactions. Taken together, our results showed that the wild-type OYE 1 reduces carvone from a less preferred substrate binding orientation. The indole ring of Trp 116 physically blocks access to a hydrophobic active site pocket. Relieving the steric congestion by mutating Trp 116 allows entry of the isopropenyl side-chain of carvone into this hydrophobic pocket and also makes the opposite face of the π system accessible to hydride addition, thereby yielding the opposite diastereomer after net trans-addition of H2.Keywords: alkene reductase; binding orientation; carvone; ene reductase; mutant; X-ray crystallography

Structural and Catalytic Characterization of Pichia stipitis OYE 2.6, a Useful Biocatalyst for Asymmetric Alkene Reductions

Co-reporter:Yuri A. Pompeu;Bradford Sullivan;Adam Z. Walton

Advanced Synthesis & Catalysis 2012 Volume 354( Issue 10) pp:1949-1960

Publication Date(Web):

DOI:10.1002/adsc.201200213

Abstract

We have probed Pichia stipitis CBS 6054 Old Yellow Enzyme 2.6 (OYE 2.6) by several strategies including X-ray crystallography, ligand binding and catalytic assays using the wild-type as well as libraries of site-saturation mutants. The alkene reductase crystallized in space group P 6₃ 2 2 with unit cell dimensions of 127.1×123.4 Å and its structure was solved to 1.5 Å resolution by molecular replacement. The protein environment surrounding the flavin mononucleotide (FMN) cofactor was very similar to those of other OYE superfamily members; however, differences in the putative substrate binding site were also observed. Substrate analog complexes were analyzed by both UV-Vis titration and X-ray crystallography to provide information on possible substrate binding interactions. In addition, four active site residues were targeted for site saturation mutagenesis (Thr 35, Ile 113, His 188, His 191) and each library was tested against three representative Baylis–Hillman adducts. Thr 35 could be replaced by Ser with no change in activity; other amino acids (Ala, Cys, Leu, Met, Gln and Val) resulted in diminished catalytic efficiency. The Ile 113 replacement library yielded a range of catalytic activities, but had very little impact on stereoselectivity. Finally, the two His residues (188 and 191) were essentially intolerant of substitutions with the exception of the His 191 Asn mutant, which did show significant catalytic ability. Structural comparisons between OYE 2.6 and Saccharomyces pastorianus OYE1 suggest that the key interactions between the substrate hydroxymethyl groups and the side-chain of Thr 35 and/or Tyr 78 play an important role in making OYE 2.6 an (S)-selective alkene reductase.

Biocatalytic Reductions of Baylis–Hillman Adducts

Co-reporter:Adam Z. Walton, W. Colin Conerly, Yuri Pompeu, Bradford Sullivan, and Jon D. Stewart

ACS Catalysis 2011 Volume 1(Issue 9) pp:989

Publication Date(Web):July 19, 2011

DOI:10.1021/cs200223f

Baylis–Hillman adducts are highly useful synthetic intermediates; to enhance their value further, we sought enantiocomplementary alkene reductases to introduce chirality. Two solutions emerged: (1) a wild-type protein from Pichia stipitis (OYE 2.6), whose performance significantly outstrips that of the standard enzyme (Saccharomyces pastorianus OYE1), and (2) a series of OYE1 mutants at position 116 (Trp in the wild-type enzyme). To understand how mutations could lead to inverted enantioselectivity, we solved the X-ray crystal structure of the Trp116Ile OYE1 variant complexed with a cyclopentenone substrate. This revealed key protein–ligand interactions that control the orientation of substrate binding above the FMN cofactor.Keywords: alkene reductase; Baylis−Hillman; enantiocomplementary; enoate reductase; old yellow enzyme; Roche’s ester; X-ray crystallography;

Drug-Delivery Strategies by Using Template-Synthesized Nanotubes

Co-reporter:Jillian L. Perry;Charles R. Martin

Chemistry - A European Journal 2011 Volume 17( Issue 23) pp:6296-6302

Publication Date(Web):

DOI:10.1002/chem.201002835

Abstract

Encapsulating drugs within hollow nanotubes offers several advantages, including protection from degradation, the possibility of targeting desired locations, and drug release only under specific conditions. Template synthesis utilizes porous membranes prepared from alumina, polycarbonate, or other materials that can be dissolved under specific conditions. The method allows for great control over the lengths and diameters of nanotubes; moreover, tubes can be constructed from a wide variety of tube materials including proteins, DNA, silica, carbon, and chitosan. A number of capping strategies have been developed to seal payloads within nanotubes. Combining these advances with the ability to target and internalize nanotubes into living cells will allow these assemblies to move into the next phase of development, in vivo experiments.

Towards preparative-scale, biocatalytic alkene reductions

Co-reporter:Despina J. Bougioukou, Adam Z. Walton and Jon D. Stewart

Chemical Communications 2010 vol. 46(Issue 45) pp:8558-8560

Publication Date(Web):04 Oct 2010

DOI:10.1039/C0CC03119D

Simple strategies for using alkene reductase enzymes to produce gram-scale quantities of both (R)- and (S)-citronellal have been developed. The methodology is easily accessible to non-specialist laboratories, allowing alkene reductases to be added to the toolbox of routine synthetic transformations.

Reductions of cyclic β-keto esters by individual Saccharomyces cerevisiae dehydrogenases and a chemo-enzymatic route to (1R,2S)-2-methyl-1-cyclohexanol

Co-reporter:Santosh Kumar Padhi, Iwona A. Kaluzna, Didier Buisson, Robert Azerad, Jon D. Stewart

Tetrahedron: Asymmetry 2007 Volume 18(Issue 18) pp:2133-2138

Publication Date(Web):17 September 2007

DOI:10.1016/j.tetasy.2007.08.010

Twenty purified dehydrogenases cloned from bakers’ yeast (Saccharomyces cerevisiae) and expressed as fusion proteins with glutathione (S)-transferase were tested for their ability to reduce three homologous cyclic β-keto esters. The majority of dehydrogenases reduced ethyl 2-oxo-cyclopentanecarboxylate, yielding a pair of diastereomeric alcohols with consistent (1R)-stereochemistry. Ethyl 2-oxo-cyclohexanecarboxylate reductions afforded only cis-alcohol enantiomers. Ethyl 2-oxo-cycloheptanecarboxylate was accepted by two enzymes in the collection, and both yielded mainly the cis-(1R,2S)-alcohol. Escherichia coli cells overexpressing the YDL124w gene were used in a dynamic kinetic resolution of ethyl 2-oxo-cyclohexanecarboxylate to produce the key intermediate in a chemo-enzymatic synthesis of (1R,2S)-2-methyl-1-cyclohexanol, an important chiral building block.cis-(1S,2R) Ethyl 2-hydroxycyclohexanecarboxylateC9H16O3Ee = 80%[α]D = −27.5 (c 0.6, CHCl3)Source of chirality: enzymatic reductionAbsolute configuration: (1S,2R)(1R,2R)-2-(Hydroxymethyl)-cyclohexanolC7H14O2Ee = 80%[α]D = −32.1 (c 0.24, H2O)Source of chirality: prior enzymatic reductionAbsolute configuration: (1R,2R)((1R,2R)-2-Hydroxycyclohexyl)methyl 2,4,6-trimethyl-benzenesulfonateC16H24O4SEe = 80%[α]D = −9.5 (c 1.0, CHCl3)Source of chirality: prior enzymatic reductionAbsolute configuration: (1R,2R)(1R,2S)-2-Methyl-cyclohexanolC7H14OEe = 80%[α]D = −16.2 (c 2.12, CHCl3)Source of chirality: prior enzymatic reductionAbsolute configuration: (1R,2S)

Chemoenzymatic formal total synthesis of (−)-bestatin

Co-reporter:Brent D. Feske, Jon D. Stewart

Tetrahedron: Asymmetry 2005 Volume 16(Issue 18) pp:3124-3127

Publication Date(Web):19 September 2005

DOI:10.1016/j.tetasy.2005.08.022

A highly stereoselective, enzymatic reduction of an α-chloro-β-keto ester provided the key intermediate for a total synthesis of the α-hydroxy-β-amino acid moiety of (−)-bestatin. The reduction product was cyclized to a glycidic ester that was opened in a Ritter reaction with benzonitrile, affording a trans-oxazoline, which was hydrolyzed under acidic conditions to the target molecule.Ethyl (2R,3S)-2-chloro-3-hydroxy-4-phenylbutyrateC12H15ClO3Ee > 98%[α]D = +24 (c 0.7, CHCl3)Source of chirality: enzymatic reductionAbsolute configuration: (2R,3S)Ethyl (2S,3S)-cis-4-phenyl-2,3-oxiranebutanoateC12H14O3Ee > 98%[α]D = +37 (c 3.0, CHCl3)Source of chirality: prior enzymatic reductionAbsolute configuration: (2S,3S)(4S,5R)-4,5-Dihydro-2-phenyl-4-carboethoxy-5-benzyl-1,3-oxazoleC19H19NO3Ee > 98%[α]D = −57 (c 2.0, CHCl3)Source of chirality: prior enzymatic reductionAbsolute configuration: (4S,5R)(2S,3R)-2-Hydroxy-3-amino-4-phenylbutyric acid hydrochlorideC10H13NO3·HClEe > 98%[α]D = +23 (c 1.3, 1 M HCl)Source of chirality: prior enzymatic reductionAbsolute configuration: (2S,3R)

Regio and enantioselective reduction of t-butyl 6-chloro-3,5-dioxohexanoate with baker’s yeast

Co-reporter:Michael Wolberg, Iwona A. Kaluzna, Michael Müller, Jon D. Stewart

Tetrahedron: Asymmetry 2004 Volume 15(Issue 18) pp:2825-2828

Publication Date(Web):20 September 2004

DOI:10.1016/j.tetasy.2004.07.045

Whole baker’s yeast cells reduce t-butyl 6-chloro-3,5-dioxohexanoate regioselectively to the corresponding C5 hydroxy keto ester. While the (R)-alcohol was favored, the enantioselectivity was poor (41% ee). A variety of process conditions were evaluated in order to improve both the enantioselectivity and yield of this reduction. Including a nonpolar resin in the reaction mixture afforded the (R)-alcohol in 94% ee and 50% isolated yield. The enantioselectivity was further improved to >99% ee by substituting purified YGL157w in place of whole yeast cells. This reductase was identified by screening a collection of yeast enzymes uncovered by genome sequence analysis.t-Butyl (R)-6-chloro-5-hydroxy-3-oxohexanoateC10H17ClO4Ee = 94%[α]D = +22.8 (c 1.6, CHCl3)Source of chirality: enzymatic reductionAbsolute configuration (5R)

Library construction and evaluation for site saturation mutagenesis

Co-reporter:Bradford Sullivan, Adam Z. Walton, Jon D. Stewart

Enzyme and Microbial Technology (10 June 2013) Volume 53(Issue 1) pp:70-77

Publication Date(Web):10 June 2013

DOI:10.1016/j.enzmictec.2013.02.012

We developed a method for creating and evaluating site-saturation libraries that consistently yields an average of 27.4 ± 3.0 codons of the 32 possible within a pool of 95 transformants. This was verified by sequencing 95 members from 11 independent libraries within the gene encoding alkene reductase OYE 2.6 from Pichia stipitis. Correct PCR primer design as well as a variety of factors that increase transformation efficiency were critical contributors to the method's overall success. We also developed a quantitative analysis of library quality (Q-values) that defines library degeneracy. Q-values can be calculated from standard fluorescence sequencing data (capillary electropherograms) and the degeneracy predicted from an early stage of library construction (pooled plasmids from the initial transformation) closely matched that observed after ca. 1000 library members were sequenced. Based on this experience, we suggest that this analysis can be a useful guide when applying our optimized protocol to new systems, allowing one to focus only on good-quality libraries and reject substandard libraries at an early stage. This advantage is particularly important when lower-throughput screening techniques such as chiral-phase GC must be employed to identify protein variants with desirable properties, e.g., altered stereoselectivities or when multiple codons are targeted for simultaneous randomization.Highlights► A site-saturation mutagenesis protocol yielding 27.4 ± 3.0/32 codons in a 95-member library was developed. ► Critical parameters impacting library quality were uncovered and optimized. ► A quantitative method correlating early-stage DNA sequencing data with library quality is described.

Towards preparative-scale, biocatalytic alkene reductions

Co-reporter:Despina J. Bougioukou, Adam Z. Walton and Jon D. Stewart

Chemical Communications 2010 - vol. 46(Issue 45) pp:NaN8560-8560

Publication Date(Web):2010/10/04

DOI:10.1039/C0CC03119D